Gluconic acid, in which the aldehyde group of D-glucose has been oxidized, and glucuronic acid, in which the hydroxymethyl group of D-glucose has been oxidized, are known as typical oxides of D-glucose. Gluconic acid and the lactone thereof, which is gluconolactone, are formed by the oxidative fermentation (gluconic acid fermentation) of glucose by a microorganism. Microorganisms such as Aspergillus niger and Penicillium chrysogenum are used in the industrial production of these compounds.
Although various methods are known for producing glucuronic acid and the lactone form thereof, which is gluconolactone, one method that has actually been industrialized is a process wherein a glucose derivative such as starch is selectively oxidized using a nitrogen oxide compound such as nitric acid and thereby converted to a carboxylic acid, following which the product of oxidization is hydrolyzed to give glucuronic acid and glucuronolactone (Patent Document 1). However, in this process, the by-product gases that form during the oxidation reaction are difficult to handle, resulting in a low yield of the target substance.
The prior art also includes a method for obtaining glucuronic acid and glucuronolactone in a high yield wherein trehalose oxide is prepared by oxidizing the hydroxymethyl group of trehalose, following which the trehalose oxide is hydrolyzed to form the desired glucuronic acid and glucuronolactone (Patent Document 2). However, although this method does provide a good yield of the target substance from the starting material, it requires a large amount of oxidation catalyst such as platinum oxide, vanadium oxide or palladium, in addition to which relatively extreme conditions are needed to hydrolyze trehalose oxide, all of which results in considerable production costs.
Another method that has been described involves using an oxidation catalyst resin to which has been adsorbed an amine oxide such as 6,6-tetramethylpiperidine-N-oxyl to oxidize a glucose derivative in the presence of a halogen-containing compound (Patent Document 3). Because nitrogen oxides such as nitric acid are not used in this method, glucuronic acid and glucuronolactone can be safely and efficiently obtained. However, the glucose derivatives preferred for use in this method are the high-cost compounds methyl-α-D-glucoside and isopropyl-(α, β)-D-glucoside. Hence, as with the above-described methods, this method also entails substantial production costs.
A relatively inexpensive glucuronic acid production process that has been described involves producing a sucrose carboxylic acid from sucrose by oxidative fermentation with a microorganism, then adding a microorganism having an invertase activity and hydrolyzing the sucrose carboxylic acid so as to obtain glucuronic acid and glucuronolactone (Patent Document 4). In this process, unlike conventional chemical synthesis processes, glucuronic acid and glucuronolactone may be obtained in high yields under mild conditions. However, the need to use two different microorganisms in the series of production steps making up this process complicates the operations.
To produce glucuronic acid directly from glucose as in gluconic acid fermentation, it is necessary to specifically oxidize the hydroxymethyl group of glucose. Enzymes that are known to oxidize the hydroxymethyl group of glucose include alcohol dehydrogenase (Patent Document 5) and alcohol/aldehyde dehydrogenase (Patent Document 6). Pseudogluconobacter sp. are known to be strains having these enzymes.
Illustrative examples of these strains include Pseudogluconobacter saccharoketogenes K591s (FERM BP-1130, IFO14464), Pseudogluconobacter saccharoketogenes 12-5 (FERM BP-1129, IFO14465), Pseudogluconobacter saccharoketogenes TH14-86 (FERM BP-1128, IFO14466), Pseudogluconobacter saccharoketogenes 12-15 (FERM BP-1132, IFO14482), Pseudogluconobacter saccharoketogenes 12-4 (FERM BP-1131, IFO14483) and Pseudogluconobacter saccharoketogenes 22-3 (FERM BP-1133, IFO14484) (Patent Document 7).
However, as is explained in detail in the subsequently described working examples, these strains have a low specificity for oxidation of the hydroxymethyl group of glucose, and form glucose oxides other than glucuronic acid, such as gluconic acid and 2-ketogluconic acid. That is, up until now, in the field of the present art, microorganisms suitable for glucuronic acid fermentation in which glucuronic acid is produced directly from glucose in the same way as in gluconic acid fermentation, and methods for producing glucuronic acid and/or glucuronolactone by glucuronic acid fermentation using such microorganisms have yet to be established.
Patent Document 1: Japanese Examined Patent Publication No. S43-5882
Patent Document 2: Japanese Laid-open Patent Publication No. H10-251263
Patent Document 3: Japanese Laid-open Patent Publication No. H11-147043
Patent Document 4: Japanese Laid-open Patent Publication No. 2006-314223
Patent Document 5: Japanese Laid-open Patent Publication No. H5-68542
Patent Document 6: Japanese Laid-open Patent Publication No. 2003-159079
Patent Document 7: Japanese Laid-open Patent Publication No. H6-7157
In light of these circumstances, the inventors have reflected on the above prior art and conducted repeated and extensive investigations with the aims of producing glucuronic acid directly from glucose in the same way as in gluconic acid fermentation as well as developing microorganisms which have a high specificity for oxidation of the hydroxymethyl group of glucose and are suitable for glucuronic acid fermentation, and of developing a new method for producing glucuronic acid and/or glucuronolactone that utilizes such microorganisms. As a result, the inventors have discovered novel mutant strains having the ability to specifically oxidize the hydroxymethyl group of glucose, and have also succeeded in establishing a new method for producing glucuronic acid and/or glucuronolactone using the mutant strains or treated forms of the cells thereof.